Let there be light and muscle contraction: optogenetic restoration of muscle function.

Abstract

Spinal cord injury is a devastating condition that greatly affects patients, their support system, and the entire healthcare system. The total economic burden to healthcare systems approaches $10 billion annually and stems from patients’ primary functional motor disability and secondary complications such as pain, weakness, and fatigue, which are closely linked to decreased social and mental health functioning. Although the use of embryonic stem cells to replace lost motor neurons within the central nervous system has been shown to engraft into a peripheral nerve environment and successfully reinnervate denervated muscle, embryonic stem cells are typically not controlled by descending inputs from the central nervous system. This leads to their neural activity being very imprecise, creating the need for more refined activation control. Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By expression of light-sensitive microbial membrane proteins (opsins), cell type–specific depolarization or silencing can be optically induced on a millisecond time scale. To that end, Bryson et al1 genetically engineered mouse embryonic stem cells to stably express channelrhodopsin-2 (ChR2), a cation channel sensitive to blue light, and glial-derived neurotrophic factor, a neurotrophic factor that promotes long-term motor neuron survival. They and then differentiated these cells in vitro to obtain optogenetically activatable ChR2 motor neurons. They then grafted aggregates of stem cells (embryoid bodies) containing these ChR2 motor neurons into a mouse model of muscle denervation in which the sciatic nerve was ligated. The engrafted ChR2 motor neurons survived, matured, and grew to innervate the denervated muscles of the lower limb. These engrafted ChR2 motor neuron axons were mostly myelinated, and histological analysis revealed robust reinnervation of muscle fibers by ChR2 motor neurons, although the neuromuscular junctions exhibited hallmarks of inactivity, most likely because these motor neurons were inactive in vivo until stimulated by the external optical signal. The ChR2 motor neurons were selectively activated by 470-nm light in a controlled manner to produce graded muscle contractions in a highly reproducible manner (Figure). Remarkably, optogenetic stimulation of ChR2 motor neurons results in motor unit recruitment patterns that closely track physiological motor unit recruitment order, unlike electric stimulation, which produces a reverse or random recruitment order. Thus, Bryson et al were able to restore muscle function through illumination of the graft site in anesthetized mice with blue light.Figure: Restoration of muscle function in a controlled manner through the use of optical stimulation of engrafted channelrhodopsin-2 (ChR2) motor neurons in vivo.1 A, schematic of optical stimulation and isometric muscle tension recordings setup. Representative twitch (B), tetanic (C), and repetitive tetanic (D) contraction traces obtained from the transversus abdominis muscle, induced by optical stimulation. Blue shows muscle force; red, light-emitting diode (LED) light triggers. E, quantification of twitch and tetanic contraction of transversus abdominis and extensor digitorum longus muscles. Time to peak contractile force, from initiation of the electrical trigger to the LED unit (F) or from the initiation of muscle contraction (G), is shown alongside direct electric nerve stimulation. H, representative fatigue traces from transversus abdominis muscles produced by optical (top) or electrical (bottom) stimulation for 180 seconds. I, representative transversus abdominis muscle optical stimulation motor-unit number estimate trace. The asterisk indicates square-wave trigger voltage to the LED unit and oscilloscope trigger. J, motor-unit number quantification of transversus abdominis and extensor digitorum longus muscles after optical versus electrical stimulation. K, analysis of average motor-unit force. The dashed line indicates the normal extensor digitorum longus value. All error bars indicate SEM. Reprinted with permission from Bryson JB, Machado CB, Crossley M, et al. Optical control of muscle function by transplantation of stem-cell derived motor neurons in mice. Science. 2014;344(6179):94-97.Clearly, many challenges remain before this approach can be established as an effective clinical intervention. A permanent, implantable optical stimulator must be developed. In addition, the long-term survival of engrafted ChR2 motor neurons must be investigated. Finally, the extension of optogenetic techniques beyond murine models to nonhuman primates must be realized. Nonetheless, this proof-of-principle study is an elegant step along the path to optogenetic translation.